Amomi Fructus, a mature fruit from a ginger family plant, has various species, resulting in inconsistent sourcing and quality. Most studies distinguish species by volatile compounds, yet research shows it also contains flavonoids with notable pharmacological effects. Solely focusing on volatile compounds could lead to considerable resource waste. This study aims to establish flavonoid markers in Amomi Fructus to distinguish its species, assess quality, and promote efficient resource use. Utilizing natural deep eutectic solvents (NADES) and response surface methodology (RSM), an optimal extraction system (choline chloride-ethylene glycol) yielded 41.38 mg RE/g total flavonoids. LC-MS analysis of 18 Amomi Fructus batches identified 26 flavonoids, quantified 19, and highlighted three key markers-epicatechin, procyanidin B2, and procyanidin B4-that effectively differentiate Amomum villosum Lour. (AMV) from Amomum villosum Lour. var. xanthioides T.L. Wu et Senjen (AMVX). Finally, flow cytometry confirmed these markers' antioxidant activity, effectively reducing H₂O₂-induced oxidative damage in GES-1 cells.